ACh receptors link two signaling pathways to neuroprotection against glutamate-induced excitotoxicity in isolated RGCs.
ACh receptors link two signaling pathways to neuroprotection against glutamate-induced excitotoxicity in isolated RGCs.
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DOI：
10.1111/j.1471-4159.2009.06447.x
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发表时间：
2010-01
影响因子：
4.7
通讯作者：
Linn CL
中科院分区：
文献类型：
--
作者：
Asomugha CO;Linn DM;Linn CL
先前的研究已经报道，激活培养的猪视网膜神经节细胞（RGC）上的烟碱型ACh受体对谷氨酸诱导的兴奋性毒性具有神经保护作用。然而，将nAChRs与神经保护联系起来的机制尚不清楚。在这里，我们测试的假设，涉及p38 MAP激酶和PI 3激酶→ Akt的信号级联参与连接激活的nAChR的神经保护在分离的猪RGC。在ELISA研究中，在存在和不存在p38 MAP激酶和PI 3激酶的特异性抑制剂的情况下诱导兴奋性毒性或神经保护后，分析磷酸化p38 MAP激酶和Akt的调节。ELISA结果显示ACh可显著增加Akt磷酸化水平，降低p38 MAP激酶活性。谷氨酸增加磷酸化p38 MAP激酶，但对磷酸化Akt没有显着影响。使用p38 MAP激酶和PI 3激酶抑制剂的其他ELISA研究也支持ACh上调PI 3激酶和Akt下游的Bcl-2水平，而谷氨酸下调p38 MAP激酶下游的Bcl-2水平的假设。随后通过在存在或不存在p38 MAP激酶或PI 3激酶的特异性抑制剂的情况下在诱导兴奋性毒性或神经保护的条件下培养RGC来评估RGC存活。p38 MAP激酶抑制剂显着降低了谷氨酸诱导的兴奋性毒性死亡的RGCs的数量，但对由于乙酰胆碱诱导的神经保护而存活的细胞数量没有影响。PI 3激酶抑制剂显着降低乙酰胆碱诱导的神经保护引起的细胞存活，但对谷氨酸诱导的兴奋性毒性引起的细胞死亡没有影响。这些结果表明，谷氨酸通过p38 MAP激酶信号通路介导兴奋性毒性，ACh通过刺激PI 3激酶→ Akt → Bcl-2信号通路和抑制p38 MAP激酶→ Bcl-2通路提供神经保护。
Previous studies have reported that activation of nicotinic ACh receptors on cultured pig retinal ganglion cells (RGCs) has a neuroprotective effect against glutamate-induced excitotoxicity. However, the mechanism linking nAChRs to neuroprotection is unknown. Here we tested the hypothesis that signaling cascades involving p38 MAP kinase and PI3 kinase → Akt are involved in linking activation of nAChRs to neuroprotection in isolated pig RGCs. In ELISA studies, regulation of phosphorylated p38 MAP kinase and Akt were analyzed after inducing excitotoxicity or neuroprotection in the presence and absence of specific inhibitors for p38 MAP kinase and PI3 kinase. ELISA results demonstrated that ACh significantly increased phosphorylated Akt and decreased p38 MAP kinase. Glutamate increased phosphorylated p38 MAP kinase but had no significant effect on phosphorylated Akt. Other ELISA studies using p38 MAP kinase and PI3 kinase inhibitors also supported the hypothesis that ACh up-regulated Bcl-2 levels downstream from PI3 kinase and Akt, whereas glutamate down-regulated Bcl-2 levels downstream from p38 MAP kinase. RGC survival was subsequently assessed by culturing RGCs in conditions to induce excitotoxicity or neuroprotection in the presence or absence of specific inhibitors of p38 MAP kinase or PI3 kinase. The p38 MAP kinase inhibitor significantly decreased the number of RGCs that died by glutamate-induced excitotoxicity but had no effect on the number of cells that survived due to ACh-induced neuroprotection. PI3 kinase inhibitors significantly decreased cell survival caused by ACh-induced neuroprotection but had no effect on cell death caused by glutamate-induced excitotoxicity. These results demonstrate that glutamate mediates excitotoxicity through the p38 MAP kinase signaling pathway and that ACh provides neuroprotection by stimulating the PI3 kinase → Akt → Bcl-2 signaling pathway and inhibiting the p38 MAP kinase → Bcl-2 pathway.
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